Current evidence indicates that many aspects of gene function are coordinated by changes in the epigenome, which includes dynamic revisions of chromatin modifications, genome packaging, subnuclear localization, and chromosome conformation. All of these epigenetic mechanisms are used by developing lymphocytes to regulate the production of functional antigen receptor genes. The genes encoding immunoglobulin (Ig) and T cell receptor (TCR) proteins are assembled by precursor lymphocytes using the process of V(D)J recombination, which generates enormous receptor diversity. These genomic rearrangements must be tightly regulated to ensure proper lymphocyte development and to avoid chromosomal translocations that cause lymphoid tumors. V(D)J recombination is controlled by a complex interplay between genetic control elements that modify the accessibility of chromatin at Ig and Tcr loci to a single V(D)J recombinase complex. Studies in the applicant's laboratory demonstrate that activation of Tcrb gene assembly requires the physical interaction between a transcriptional enhancer (E2) and promoters (PD2), which form a stable holocomplex in thymocytes. The PD2/E2 holocomplex opens chromatin associated with closely-spaced D2 and J2 gene segments, providing access to recombinase and promoting the first step in Tcrb gene assembly (D2`J2 recombination). In contrast to initial Tcrb activation, very little is known about the genetic and epigenetic control of long-range rearrangement events that must occur between V2 gene segments and rearranged D2J2 elements, located more than 250 kb away. Emerging studies indicate that the Tcrb locus contracts in thymocytes to bring V2 gene segments into spatial proximity of the D2J2 cluster. The central hypothesis of the proposed research project is that a series of known and novel genetic control elements mediate the opening of V2 chromatin and facilitate interactions between this large cluster of gene segments and the distant D2J2 region. To test this hypothesis, the applicant proposes to (i) determine whether known regulatory elements in the D2J2 region are essential for Tcrb locus contraction in primary thymocytes and (ii) identify novel genetic control elements within the V2 cluster that activate transcription of the composite gene segments and coordinate their long-range interactions with the D2J2 region. Together, these studies will provide a new paradigm for the chromosomal dynamics that guide antigen receptor gene assembly and prevent aberrant interactions that may lead to oncogenic translocations with other chromosomes. PUBLIC HEALTH RELEVANCE: The packaging and spatial organization of DNA in the nucleus are key determinants of gene function;changes in these "epigenetic" programs may be at the heart of aging and disease susceptibilities. In addition, epigenetic control is essential for the proper generation of antigen receptor genes because defects in this assembly process cause chromosomal translocations that lead to lymphoid tumors. We propose studies to address how precursor T lymphocytes control long-range changes in the spatial organization of T cell receptor beta loci to ensure their proper assembly and pursuant generation of a normal adaptive immune system.